PROJECT SUMMARY/ABSTRACT Misfolding and aggregation of proteins is a central pathogenic event in neurodegenerative disorders such as Alzheimer?s (AD) and Parkinson?s (PD) diseases and one that is believed to occur well before, perhaps by decades, the onset of neurodegeneration and cognitive impairment. Monitoring the course of protein aggregation at the biochemical, cellular, and tissue levels is thus vital to understanding and combating these diseases. Currently, there lacks useful sensors for detecting the range of protein aggregates involved in disease etiology, particularly the oligomeric and pre-fibrillar aggregate conformations that are the most neurotoxic. Moreover, useful sensors need to simultaneously detect aggregates formed by different proteins as comorbid pathologies caused by the co-deposition of multiple proteins is characteristic of a number of diseases. We have recently demonstrated that a class of sensors based on a novel molecular scaffold, oligomeric p-phenylene ethynylene (PE)-based electrolytes (OPEs), has vastly expanded and superior sensing capability. In vitro, OPEs selectively detected synthetic fibrils of two model proteins and eight variants of AD-related Ab and tau and PD-related a- synuclein proteins over their monomeric counterparts. Compared to the gold-standard thioflavin T dye, the small anionic OPE1- exhibited higher selectivity and sensitivity at detecting amyloid fibrils. OPE1- also detected a- synuclein oligomers and fibrils that thioflavin T failed to detect. Collectively, OPEs promise significant advancement in the detection of the myriad of protein aggregates involved in the early stages of AD and PD. further develop the use of these new probes, we propose to test and validate OPE sensing in three biological systems with increasing complexity, in vitro sensing of Ab, tau, and a-synuclein aggregates isolated from AD and PD transgenic mice brains and postmortem AD and PD human brain tissue (Aim 1), ex vivo staining of protein aggregates in transgenic mice and human brain sections (Aim 2), and in vivo OPE sensing of protein aggregates in transgenic mice models (Aim 3). We propose to perform live brain two-photon imaging on transgenic and wildtype control mice after tail-vain injection of OPE. Blood-brain-barrier (BBB) permeability and biodistribution of OPEs will additionally be investigated. Our goal is to acquire preliminary, in vivo feasibility data for an R01 application for further probe development and expanded in vivo testing. The long-term goal of our project is to provide researchers and clinicians with robust and facile protein aggregate detection agents to investigate the link between pre-clinical pathophysiological processes and later emergence of clinical symptoms. Achievement of our study aims will create a crucial ?pre-clinical? window of opportunity to study and intervene with disease-modifying therapy.